Mecanismos moleculares de resistência em enterobactérias e avaliação de uma nova abordagem terapêutica

Abstract

The increase of hospital infections caused by multidrug-resistant Enterobacteriaceae is a major challenge for Public Health and a main problem for the treatment of patients hospitalized in Intensive Care Units (ICUs). The identification of genetic mechanisms involved in bacterial resistance and the search for new therapies have major implications in the reduction and control of multidrug-resistant strains. The objective of this study was to characterize the resistance mechanisms to polymyxin in Enterobacteriaceae involved in outbreaks by genome sequencing, associating clinical and epidemiological data of the patients. In addition, the study evaluated the antimicrobial activity of an antisense peptide against a multidrug-resistant strain, seeking to contribute in the control of the dissemination of these strains. During September/2015 to February/2017, 53 strains of Enterobacteriaceae with polymyxin-resistance profile were isolated from two Brazilian hospitals. Of these strains, 30 were identified as Klebsiella pneumoniae and submitted to genome sequencing. The results revealed that the blaKPC gene was the main resistance gene identified, followed by blaSHV, blaTEM, blaCTX-M and blaOXA. Other genes that confer resistance to aminoglycosides, fluoroquinolones, sulfonamides, tetracyclines, trimethoprim and macrolides were also detected. All isolates showed high genetic similarity and polymyxin resistance was attributed to three mechanisms involving inactivation of the mgrB gene, including inactivation by insertion sequences (IS) and point mutations. An 80 bp insertion sequence was identified in six strains which results in duplication of 26 amino acids of the MgrB protein. This is the first observation of this type of change causing polymyxin resistance. The characterization of polymyxin resistance was also performed in Klebsiella aerogenes isolates, in this study nine strains were isolated from patients admitted to ICUs. A clonal expansion of the isolates was observed and the genome sequencing data revealed that strains were clustered into two clonal clusters. Genomic analysis showed that polymyxin resistance was mediated by three distinct mutational mechanisms, including nonsense point changes and amino acid substitution in PhoP and PhoQ proteins. In addition, we identify an unusual mutation in the soxS gene resulting in a truncated protein. A case-control study was conducted with 159 patients to explore the potential risk factors associated with the acquisition of polymyxin-resistant strains producing carbapenemases isolated from patients in adult and neonatal ICUs, as well as to describe the mortality and clinical characteristics of these patients’ infections. In adult patients, several risk factors were identified, including renal failure, urinary catheter use, surgical procedures, exposure to carbapenem and transfer between hospital wards. In neonates, use of central venous catheter was the main risk factor identified. Mortality was significantly higher in patients infected with polymyxin-resistant strains than in those with polymyxin-sensitive strains (p<0.01). The study of risk factors also showed that use of mechanical ventilation and polymyxin exposure were strongly associated with the mortality of patients included in the study. In addition, the antimicrobial potential of an antisense peptide (PNA) against KPC-producing K. pneumoniae was investigated in vitro. PNA inhibited bacterial growth at 50 μM, with a 96.7% reduction in 16s gene amplification. PNA showed low hemolytic activity at concentrations necessary to kill bacteria and bioinformatics analyzes demonstrated that the PNA structure exhibits good conformational stability in biological fluids. Based on these results, it is concluded that the identification of the genetic mechanisms involved in antimicrobial resistance and the identification of specific risk factors are important in the search for innovative strategies for the control of these strains, as well as for the development of new therapeutic approaches for the treatment of these infections.